Quantitative RT-PCR analysis ABA- and SA-treated leaf tissues

Wheat seedlings (cv. Morocco) were grown in a controlled growth chamber environment (16-h light, 8-h dark, 22 °C) for fourteen days until the 2-leaf development stage. Three biological replicates were generated for each of four foliar applications: mock (aqueous solution alone), abscisic acid (2 mM), and salicylic acid (50 mM and 100 mM). Treatments were all applied as foliar sprays in an aqueous solution (40% Ethanol, 0.05% TritonX-100 + treatment) until leaves were dripping. About 100 mg of leaf tissue was collected from each application 18 h after treatment. Total RNA was extracted using Trizol reagent (Life Technologies) according to manufacturer’s protocol. Each RNA sample was treated with DNase I (Life Technologies) according to manufacturer’s protocols. DNase-treated RNA from each biological replicate was used to generate cDNA for each sample.

Samples from each biological replicate were tested by mixing four technical replicates of the same qPCR reaction mixture using the IQ SYBR green super mix reaction mixture (BIO-Rad) with one set of experimental primers specific for POX2 (5′- GTCATACTGCAGCCTGTTGCCTTC-3′, 5′- GCTTCCCAACTCTACCTAGCTGGATAC-3′), MYBa (5′- GGTGATGGCAGCAGAGGG-3′, 5′- GGCGAGCAGGAACTTCATGGTG-3′), MYBb (5′- CATGAGCCCACTTGGAATGCTAGATAG-3′, 5′- GAGGCAGGCTGGAAGATGGATGAG-3′), and MYBd (5′- GGTGATGGTAGCAGAGGACCAGAG-3′, 5′-ATTCAGCCACAGACGCCATCG-3′) or the house keeping actin primer set (5′- ACCTTCAGTTGCCCAGCAA-3′, 5′- CAGAGTCGAGCACAATACCAGTTG-3′). Each experimental primer set was paired with four technical qPCR replicates of the actin primer. Reactions were carried out on a CFX96 Real-Time system (Bio-Rad) with the same thermal cycler protocol consisting of a one-time 95 °C − 3 min, followed by 40 cycles 95 °C − 15 sec, 60 °C − 30 sec, 72 °C − 40 sec, followed by automatic dissociation analysis to asses primer specificity. Raw fluorescent quantification results for each cycle were used for normalization and to calculate relative transcript abundance using the online Real Time PCR Miner v. 4.0 [39].